The Intracellular Traffic Of Mt1-mmp Is Regulated By ¦Á2-macroglobulin/lrp1 System In m¨¹Ller Glial Cell.

BARCELONA P; JALDIN-FINCATI J; CHIABRANDO GA; SANCHEZ MC

Congreso; ARVO Meeting 2012; 2012

Purpose: M¨¹ller cells (MC) are known to undergo functional and morphological changes related with the matrix metalloproteinases (MMPs) production during retinal proliferative disorders. Previously, we demonstrated that MC express the alpha2-Macroglobulin (¦Á2M) receptor, LDL receptor-related protein 1 (LRP1), and that under ¦Á2M treatment, LRP1 regulates the MMP-2 activity and cell migration on different matrix-protein-coated surfaces, which suggest the participation of MT1-MMP. Herein, we evaluated whether LRP1 regulates the MT1-MMP function in MC stimulated by ¦Á2M. In addition, we investigated the subcellular distribution of MT1-MMP and LRP1 and evaluated the cell migration induced by ¦Á2M on different matrix-protein-coated surfaces.Methods: Transient transfection of the MIO-M1 cells with MT1-MMP-GFP vector was performed using Lipofectamine 2000. These cells cultured in Dulbecco¡¯s modified Eagle¡¯s medium (DMEM) were stimulated with ¦Á2M (60 nM) for different times. The cellular distribution of MT1-MMP and LRP1 was examined by confocal microscopy using GFP-conjugated MT1-MMP, specific antibodies against LRP1 and intracellular compartments of endocytosis. The molecular association of MT1-MMP/LRP1 was analyzed by immunoprecipitation (IP). The protein silencing of LRP1 was performed using siRNA LRP1 with siPORT Neo FX Transfection agent.Results: MIO-M1 cells, under ¦Á2M treatment, showed that LRP1 and MT1-MMP were mainly co-localized in endosomal compartments characterized as EEA1 early endosomes. By IP assay we showed a molecular association between MT1-MMP and LRP1, which was increased by ¦Á2M stimulation. Finally, the LRP1 silencing abrogated the MMP2 activation and cell migration capacity, promoted by ¦Á2M, in this cells. Conclusions: These data demonstrated that MT1-MMP is associated with LRP1 at intracellular level in MIO-M1 cells stimulated with ¦Á2M, which suggest that this receptor is regulating the intracellular traffic of MT1-MMP. In addition, the MT1-MMP/LRP1 interaction induced by ¦Á2M, regulates the MMP-2 activation and cellular migration of MC.